Cas9 (CRISPR associated protein 9) is an RNA-guided DNA nuclease associated with the Type II bacterial CRISPR immunity system. Cas9 can be used in vitro and in vivo to induce site-specific double stranded breaks in DNA. In most cases, the RNA guide structure that binds and directs Cas9 is a hybrid of CRISPR RNA, containing homologous sequence to the cleavage target, and a trans-activating “tracr” RNA. A single guide RNA “sgRNA” chimera of the CRISPR (Cr) and tracr RNAs can direct DNA cleavage by Cas9 in vitro.